이 작업은 이스라엘 무역 노동부의 KAMIN 프로그램과 Qrons Inc., 777 Brickell Avenue Miami, FL 33131, US에서 자금을 지원했습니다.

이 프로토콜에서 동물의 사용은 국가 동물 관리 및 사용위원회의 승인을 받았습니다.
참고: 탄산칼슘 산호 골격은 아라고나이트의 결정 형태로 사용해야 합니다. 지금까지 신경 배양에 대해 테스트 된 산호 유형은 Porites Lutea, Stylophora Pistillata 및 Trachyphyllia Geoffroyi입니다. 해골은 전체 또는 지상으로 구입할 수 있습니다.
1. 산호 골격 조각 청소
주의 : 아래 설명된 용액은 위험하고 화상과 자극을 유발할 수 있으므로 실온의 화학 후드에서 다음 단계를 수행해야 합니다.
<ol><li>망치를 사용하여 산호 골격을 부수고 0.5-2cm 조각으로 나눕니다. 유기 및 비 유기 잔류 물을 용해시키기 위해 산호 골격 조각을 10 % 차아 염소산 나트륨 용액에 10 분 동안 담근 다음 이중 증류수 (DDW)로 한 번 씻으십시오.</li>
	<li>남아있는 유기 잔류 물을 제거하려면 1M NaOH 용액에 5 분 동안 담근 다음 DDW로 한 번 씻으십시오. 단편을 30%H2O2용액에 10분 동안 담궈 유기 침전물의 제거를 계속한 다음, 단편을 DDW로 3x 세척한다.</li>
	<li>가능한 한 많은 DDW를 제거하고 후드의 산호 조각을 건조시킵니다 (1-8 시간).</li>
</ol>2. 유리 커버슬립 청소
<ol><li>유리 커버슬립을 100mm 유리 페트리 접시에 옮깁니다. 95% 에탄올 10mL를 15분 동안 넣습니다.</li>
	<li>에탄올을 제거하고 DDW 3x로 세척하고 각 세척 사이에 10 분을 기다립니다. DDW가 증발할 때까지 80°C로 예열된 가열판에 접시를 놓습니다. 건조하는 동안 플레이트 안의 커버슬립이 서로 달라붙지 않도록 여러 번 부드럽게 저어줍니다.</li>
	<li>커버슬립을 오토클레이브합니다.</li>
</ol>3. 산호 골격 알갱이의 제조
<ol><li>산호 골격 조각을 박격포와 유봉 (수동 분쇄)을 사용하여 완전히 분해 될 때까지 분쇄하십시오. 결과는 20 μm-200 μm 범위의 입자 혼합물입니다.</li>
	<li>또는 전기 연삭기를 사용하여 30 초 동안 1,000rpm의 속도로 산호 골격 조각을 분쇄합니다 (블레이드 길이 = 6cm, 너비 = 0.5cm-1.0cm). 생성 된 입자 크기는 수동 분쇄에 의해 생성 된 크기 범위와 유사합니다.</li>
</ol>4. 특정 크기 범위의 입자 정제
알림: 매트릭스의 입자 크기를 제어하려는 경우 다음 여과 기반 입자 정제 절차를 사용하십시오.
<ol><li>곡물을 수동 또는 전기식 40μm 필터 메쉬 스트레이너로 옮깁니다.</li>
	<li>스트레이너를 통해 곡물을 체질하여 곡물을 두 개의 특정 범위로 나눕니다(전기 여과기를 사용하는 경우 다음 조건이 권장됩니다: 흔들림 = 600amplitudes/min, 바운싱 = 6/s). 이 절차는 입자 크기의 두 그룹, 즉 하나는 <40μm이고 두 번째는 >40μm입니다. 알림: 두 가지 크기는 다양한 메쉬의 스트레이너를 사용하여 결정할 수 있습니다. 더 제한된 범위가 필요한 경우 4.2 단계에서 다른 메쉬를 가진 스트레이너를 통해 각 그룹을 다시 체로 만듭니다.</li>
	<li>곡물을 고압 증기 멸균하십시오.</li>
</ol>5. 산호 곡물 코팅 접시 또는 커버 슬립의 준비
<ol><li>플라스크, 접시 또는 페트리 접시 코팅용 알림: 다음 단계는 멸균 조건에서 수행해야 합니다.<ol><li>행크스 용액에 용해 된 20 μg / mL 폴리 -D- 라이신 (PDL) 용액에 산호 골격 알갱이를 첨가합니다. 권장 농도는 PDL 용액 1mL당 곡물 5mg/10mg입니다.</li>
		<li>용액을 플라스크와 접시(약 2mL/25cm2)에 붓고 4°C에서 밤새 배양합니다. 곡물은 가라 앉고 바닥에 붙어 있습니다.</li>
		<li>다음날, 플라스크와 접시를 멸균 DDW로 한 번 씻으십시오. 플라스크와 접시를 후드에서 말리십시오. 알림: 갓 코팅된 플라스크와 접시를 사용하는 것이 좋습니다. 코팅된 플라스크 및 접시는 4°C에서 보존하면 코팅 후 최대 일주일까지 사용할 수 있습니다. 그러나 매트릭스의 효율성이 저하 될 수 있습니다.</li>
	</ol></li>
	<li>코팅 유리 커버슬립(직경 12mm, 두께 0.17mm)<ol><li>원하는 농도로 DDW에 산호 골격 알갱이를 추가하십시오. 신경 세포에 사용되는 일반적인 밀도는 5mg/mL 및 10mg/mL입니다. 40μL의 곡물 용액을 커버슬립 중앙에 붓습니다(그림 4).</li>
		<li>80°C로 예열된 가열판에 커버슬립을 놓고 완전히 증발할 때까지 기다립니다(보통 15분). 이러한 조건 하에서, 곡물은 커버 슬립에 부착됩니다. 코팅된 커버슬립을 오토클레이브합니다. 멸균 상태에서 보관하십시오.</li>
		<li>배양 하루 전, 멸균 된 24 웰 플레이트의 뚜껑에 커버 슬립을 놓습니다. 각 커버슬립에 20μg/mL PDL 용액 100μL를 추가합니다. 팁을 사용하여 액체가 전체 입자 영역과 나머지 커버슬립 표면을 덮도록 합니다.</li>
		<li>접시 바닥으로 뚜껑을 덮으십시오. 플레이트의 측면을 파라핀 필름으로 감 쌉니다. 4°C에서 하룻밤 동안 배양합니다.</li>
		<li>다음날 DDW로 한 번 씻고 후드에서 말립니다. 알림: 갓 코팅된 커버슬립을 사용하는 것이 좋습니다.</li>
	</ol></li>
</ol>6. 산호 골격 곡물 코팅 유리 커버 슬립에서 해마 해리 세포의 배양
참고: 해마 해리 세포 배양 방법은 이전에 발표된 절차24,27에서 수정되었습니다. 배양 물의 준비는 4 마리의 쥐 새끼에 대해 설명된다. 각 해마의 예상 수율은 1-1.5 x 106 세포입니다.
<ol><li>설치<ol><li>빈 60mm 페트리 접시를 준비합니다. 최소 필수 배지(MEM) 2mL를 60mm 페트리 접시에 붓고 얼음 위에 올려 놓습니다.</li>
		<li>MEM 2mL를 35mm 접시에 붓고 37°C, 5–10%CO2의 인큐베이터에 넣습니다. 2mL의 MEM을 15mL 튜브에 붓고 4°C에서 유지합니다.</li>
		<li>First Day Medium 1mL를 15mL 튜브에 붓고 37°C, 5-10%CO2의 인큐베이터에 넣는다. 참고: First Day Medium의 구성은 재료 표에 설명되어 있습니다.</li>
		<li>2.5% 트립신 용액 200μL를 해동하고 37°C에서 배양합니다.</li>
		<li>화염을 사용하여 직경 0.5mm, 0.75mm 및 1.0mm 헤드가 있는 유리 파스퇴르 피펫 3개를 준비합니다.</li>
		<li>적절한 수술 도구를 준비하십시오 : 큰 가위 1 개, 작은 가위 2 개, 큰 핀셋 1 개, 작은 핀셋 4 개, 메스 1 개.</li>
		<li>후드에 실체 현미경을 준비하십시오.</li>
	</ol></li>
	<li>큰 가위를 사용하여 0-3일 된 Sprague Dawley 쥐 새끼의 머리를 몸에서 분리하여 빈 60mm 페트리 접시에 넣어 희생합니다(6.1.1단계). 시체를 처리하십시오.</li>
	<li>큰 핀셋으로 입에서 머리를 잡고 들어 올리십시오. 작은 가위로 두개골을 덮고있는 피부를 자릅니다.</li>
	<li>깨끗한 가위로 두개골 아래(소뇌 측면)로 들어가 두개골을 오른쪽과 왼쪽으로 잘라 제거할 수 있도록 합니다. 작은 핀셋을 사용하여 뇌에서 두개골을 떼어냅니다.</li>
	<li>깨끗한 핀셋으로 뇌를 머리에서 분리하고 60mL의 차가운 MEM이 들어 있는 2mm 페트리 접시에 넣습니다(6.1.2단계 참조).</li>
	<li>두 개의 작은 핀셋을 사용하여 실체 현미경으로 해마를 해부합니다. 해마를 MEM이 들어있는 준비된 35mm 접시 (6.1.2 단계)에 옮깁니다. 알림: 각 강아지에 대해 6.3–6.6단계를 반복해야 합니다.</li>
	<li>메스를 사용하여 해마를 약 1mm 조각으로 자릅니다. 트립신 용액 200 μL를 첨가한다 (0.25%의 최종 농도로 희석한다). 부드럽게 혼합하고 37 °C, 5–10 % CO2 에서 30 분 동안 배양합니다.</li>
	<li>배양 후 2mL의 차가운 MEM(단계 6.1.2)을 접시에 넣어 트립신을 비활성화합니다. 트립신화된 조직을 37°C로 예열된 1 mL First Day Medium이 들어있는 15 mL 튜브로 옮기고(단계 6.1.3) 가장 큰 직경을 갖는 유리 파스퇴르 피펫을 사용한다(단계 6.1.5). 참고: 조직의 추가 처리를 위한 최상의 배지 대 조직(v:v) 비율은 1mL/8 해마입니다. 가능한 한 조직과 함께 배지를 옮기지 마십시오.</li>
	<li>조직을 가장 큰 직경의 유리 피펫에 10-15 번 통과시켜 분쇄하십시오. 그런 다음 중간 직경의 유리 피펫을 사용하여 이 과정을 반복합니다. 가장 작은 직경의 파이펫으로 연삭을 계속하십시오. 세포 사멸을 줄이기 위해 버블링을 피하십시오.</li>
	<li>나머지 조직 조각을 2-5분 동안 가라앉힌 다음 상층액을 다른 15mL 튜브로 옮깁니다. 혈구계를 사용하여 세포를 계수합니다. 참고: 바람직한 세포 밀도는 200,000–400,000 cells/mL입니다. First Day Medium을 사용하여 희석하거나 470 x g 에서 원심분리하여 세포를 농축합니다.</li>
	<li>각 유리 커버슬립에 100μL의 세포를 시드합니다. 씨를 뿌리는 동안 전체 커버 슬립을 세포로 덮으십시오. 37 °C, 5–10%CO2에서 배양합니다.</li>
	<li>다음날, 500 μL의 뉴런 성장 배지를 웰에 첨가한다. 참고: Neuronal Growth Medium의 구성은 재료 표에 설명되어 있습니다.</li>
	<li>커버슬립을 기울여 First Day Medium을 제거하면서 핀셋을 사용하여 각 커버슬립을 적절한 웰로 부드럽게 옮깁니다. 뚜껑에서 남은 매체를 흡입합니다.</li>
	<li>37 °C, 5–10%CO2 에서 배양합니다. 배양하는 동안 배지를 교체하지 마십시오. 배양 물은 이러한 조건 하에서 최대 1 개월까지 유지 될 수 있습니다. 주요 관심사는 습도입니다. 따라서 인큐베이터를 최대한 가습 상태로 유지하십시오.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+Biomaterials+and+Stem+Cells+for+Neural+Regeneration.">Integrating Biomaterials and Stem Cells for Neural Regeneration.</a> Stem Cells and Development. 25 (3), 214-226 (2016).</li><li>Drury, J. L., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+tissue+engineering:+scaffold+design+variables+and+applications.">Hydrogels for tissue engineering: scaffold design variables and applications.</a> Biomaterials. 24 (24), 4337-4351 (2003).</li><li>Woerly, S., Marchand, R., Lavall&#233;e, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+implantation+of+synthetic+polymer/biopolymer+matrix:+a+new+perspective+for+brain+repair.">Intracerebral implantation of synthetic polymer/biopolymer matrix: a new perspective for brain repair.</a> Biomaterials. 11 (2), 97-107 (1990).</li><li>Dillon, G. P., Yu, X., Sridharan, A., Ranieri, J. P., Bellamkonda, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+physical+structure+and+charge+on+neurite+extension+in+a+3D+hydrogel+scaffold.">The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold.</a> Journal of Biomaterials Science, Polymer Edition. 9 (10), 1049-1069 (1998).</li><li>Carballo-Molina, O. A., Velasco, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+scaffolds+and+delivery+systems+to+enhance+axonal+regeneration+after+injuries.">Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries.</a> Frontiers in Cellular Neuroscience. 9, (2015).</li><li>George, J., Hsu, C. C., Nguyen, L. T. B., Ye, H., Cui, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering+with+structured+hydrogels+in+CNS+models+and+therapies.">Neural tissue engineering with structured hydrogels in CNS models and therapies.</a> Biotechnology Advances. , (2019).</li><li>Shany, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aragonite+crystalline+biomatrices+support+astrocytic+tissue+formation+in+vitro+and+in+vivo.">Aragonite crystalline biomatrices support astrocytic tissue formation in vitro and in vivo.</a> Tissue Engineering. 12 (7), 1763-1773 (2006).</li><li>Baranes, D., L&#243;pez-Garc&#237;a, J. C., Chen, M., Bailey, C. H., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+hippocampal+mossy+fiber+and+associational-commissural+pathways+in+a+novel+dissociated+cell+culture+system.">Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (10), 4706-4711 (1996).</li><li>Peretz, H., Talpalar, A. E., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+survival+and+durability+of+neurons+and+astrocytes+on+3-dimensional+aragonite+biomatrices.">Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices.</a> Tissue Engineering. 13 (3), 461-472 (2007).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tissue Engineering. 11 (3-4), 585-596 (2005).</li><li>Baranes, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue Engineering. 13 (3), 473-482 (2007).</li><li>Morad, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliosis+of+astrocytes+cultivated+on+coral+skeleton+is+regulated+by+the+matrix+surface+topography.">Gliosis of astrocytes cultivated on coral skeleton is regulated by the matrix surface topography.</a> Biomedical Materials. 14 (4), 045005 (2019).</li><li>Green, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Therapeutic+Potential+for+Marine+Skeletal+Proteins+in+Bone+Regeneration.">A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration.</a> Marine Drugs. 11 (4), 1203-1220 (2013).</li><li>Neto, A. S., Ferreira, J. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+and+Marine-Derived+Porous+Scaffolds+for+Bone+Tissue+Engineering.">Synthetic and Marine-Derived Porous Scaffolds for Bone Tissue Engineering.</a> Materials. 11 (9), (2018).</li><li>Ahmad Khalili, A., Ahmad, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Cell+Adhesion+Studies+for+Biomedical+and+Biological+Applications.">A Review of Cell Adhesion Studies for Biomedical and Biological Applications.</a> International Journal of Molecular Sciences. 16 (8), 18149-18184 (2015).</li><li>. Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19218486">https://www.ncbi.nlm.nih.gov/pubmed/19218486</a> (2019)</li><li>Drake, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+skeletal+organic+matrix+from+the+stony+coral+Stylophora+pistillata.">Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (10), 3788-3793 (2013).</li><li>Ramos-Silva, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skeletal+proteome+of+the+coral+Acropora+millepora:+the+evolution+of+calcification+by+co-option+and+domain+shuffling.">The skeletal proteome of the coral Acropora millepora: the evolution of calcification by co-option and domain shuffling.</a> Molecular Biology and Evolution. 30 (9), 2099-2112 (2013).</li><li>Peretz, H., Blinder, P., Baranes, D., Vago, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aragonite+crystalline+matrix+as+an+instructive+microenvironment+for+neural+development.">Aragonite crystalline matrix as an instructive microenvironment for neural development.</a> Journal of Tissue Engineering and Regenerative Medicine. 2 (8), 463-471 (2008).</li><li>Morad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CaCO3 Matrix Dictates Astrocytes Transition to Astrogliosis. , (2019).</li><li>Prada, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocean+warming+and+acidification+synergistically+increase+coral+mortality.">Ocean warming and acidification synergistically increase coral mortality.</a> Scientific Reports. 7, (2017).</li></ol>

저자는 경쟁하는 재정적 이익이 없다고 선언합니다.

여기에 제시된 기술은 배양에서 신경 세포의 유지 및 기능을 향상시키는 방법을 설명합니다. 이것은 세포를 키우고 성장과 활동을 촉진하는 산호 골격 알갱이로 만들어진 매트릭스에 세포를 부착함으로써 달성됩니다. 이 기술을 사용하면 뇌의 세포 환경을 모방하는 신경 배양 모델의 능력이 증가합니다.
배양 기질로서 매트릭스의 도입은 고전적인 신경 세포 배양 방법에 사?...

해리된 신경 세포(이 경우 해마 세포)의 배양은 높은 세포 분리 및 접근성을 제공함으로써 신경 성장 및 기능을 연구하기 위한 귀중한 실험 모델입니다 1,2,3. 이러한 유형의 배양은 성장 및 생존 속도, 신경 독성, 신경 돌기 성장 및 네트워킹, 시냅스 연결 및 가소성, 형태 학적 변형, 신경 돌기 조직 및 배선 등과 같이 수집 할 수있는 많은 양의 정보로 인해 신경 과학, 약물 개발 및 조직 공학에서 자주 사용됩니다.1,4,5,6,7.
배양의 중요성에도 불구하고, 배양 된 세포는 일반적으로 2 차원 단층의 유리 커버 슬립에서 자라도록 강요됩니다. 이러한 엄격한 환경 변형은 시간이 지남에 따라 신경 세포의 생존 능력을 크게 감소시키는데, 이는 유리 커버슬립이 접착 강도가 낮고 세포 성장을 지원하는 능력이 낮기 때문입니다 8,9,10,11.
배양된 신경 세포는 어려운 조건에서 성장해야 하기 때문에 생존을 향상시키기 위한 필수적인 접근 방식은 가능한 한 자연 환경을 모방하는 것입니다12,13. 이것은 매트릭스 역할을 하고 세포의 세포외 기질을 모방하여 조직과 같은 구조를 형성하고 영양을 공급할 수 있도록 하는 생체 재료를 사용하여 달성할 수 있습니다14.
생체 재료의 사용은 생체 적합성 스캐폴드의 역할을 하고 기계적 안정성을 제공하며 접착, 생존, 증식, 이동, 형태 형성 및 분화를 포함한 다양한 세포 특성을 향상시키기 때문에 세포 배양을 개선하는 유망한 접근 방식입니다15,16,17. 시험관 내에서 세포의 상태를 개선하기 위해 여러 유형의 생체 재료가 사용됩니다. 그 중에는 생체 고분자 또는 일반적으로 세포의 세포 외 기질의 일부인 생물학적 성분이 있습니다. 이러한 생체 재료는 주로 중합 코팅제 또는 하이드로겔의 형태로 사용된다(18,19,20). 한편, 위에서 언급한 매트릭스는 세포가 성장할 수 있는 친숙한 3D 환경을 제공하고, 접시에 대한 접착을 촉진하며, 기계적 지지를 제공합니다(21,22). 반면에, 이들의 중합 된 형태와 하이드로 겔 내 세포의 감금은 성장 배지에 존재하는 육성 성분에 대한 세포의 접근을 방해하고 또한 미세한 방법에 의한 세포의 후속 조치를 더 어렵게 만든다23.
산호 외골격은 생물학적 해양 기원 매트릭스입니다. 탄산칼슘으로 만들어졌으며 기계적 안정성이 있으며 생분해성이 있습니다. 산호 골격을 배양에서 성장하는 신경 세포의 매트릭스로 사용한 이전 연구에서는 유리 커버 슬립에 비해 훨씬 더 큰 접착력을 보여주었습니다24,25. 또한, 산호 골격에서 자란 신경 세포는 골격을 구성하는 칼슘을 섭취하는 능력을 입증했으며, 이는 영양 결핍 상태에서 신경 세포를 보호한다26. 또한, 산호 골격은 신경 세포의 생존을 증가시키고, 신경망의 형성을 촉진하고, 시냅스 연결의 속도를 높이고, 조직과 같은 구조의 형성을 가능하게하는지지 및 양육 매트릭스입니다27,28. 최근 연구에 따르면 산호 골격 매트릭스의 표면 지형은 신경교 세포의 분포와 활성화에 중요한 역할을 합니다 8,29. 또한, 산호 골격은 배양에서 골세포30,31, 간세포 및 심근 세포와 같은 다른 세포 유형의 배양을위한 매트릭스로서 효과적이다 (미공개 데이터).
따라서 산호 골격은 시험관 내에서 세포 배양을위한 유망한 매트릭스입니다. 따라서 아래에 자세히 설명 된 프로토콜은 기존 방법으로 달성 한 것보다 더 안정적이고 번영하는 신경 배양을 생산하기 위해 산호 골격에서 신경 세포를 배양하는 기술을 설명합니다. 이 프로토콜은 또한 심근 세포, 간세포 및 다른 세포 유형의 배양에 유용 할 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Greiner</td>
			<td>#60-662160</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>#17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>#A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>D &#8211; glucose</td>
			<td>Sigma</td>
			<td>#G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Minimal Essential Eagle (DMEM)</td>
			<td>Sigma</td>
			<td>#D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical sieve</td>
			<td>Ari Levy</td>
			<td>#3700</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serun (FBS)</td>
			<td>Biological Industries</td>
			<td>#04-007-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>First Day Medium</td>
			<td></td>
			<td></td>
			<td>85.1% Minimum Essential Eagle&#8217;s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose.</td>
		</tr>
		<tr>
			<td>Flasks</td>
			<td>Greiner</td>
			<td>#60-690160</td>
			<td>25cm^2, Tissue culture treated</td>
		</tr>
		<tr>
			<td>Fluoro-deoxy-uridine</td>
			<td>Sigma</td>
			<td>#F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Menzel-Glaser</td>
			<td>#BNCB00120RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Romical</td>
			<td>#007130-72-19</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Sigma</td>
			<td>#N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>HANK&#39;S solution</td>
			<td>Sigma</td>
			<td>#H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>Sigma</td>
			<td>#K3375</td>
			<td></td>
		</tr>
		<tr>
			<td>L - glutamine</td>
			<td>Sigma</td>
			<td>#G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual strainer (40&#181;m)</td>
			<td>VWR</td>
			<td>#10199-654</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimun Essential Eagle (MEM)</td>
			<td>Sigma</td>
			<td>#M2279</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td>De-Groot</td>
			<td>4-P090</td>
			<td></td>
		</tr>
		<tr>
			<td>NaClO (Sodium Hypochlorite)</td>
			<td>Sigma</td>
			<td>#425044</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>#S8045</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Neuronal Growth Medium</td>
			<td></td>
			<td></td>
			<td>45% MEM, 40% Dulbecco&#39;s modified eagle&#39;s medium (DMEM), 10% Nutrient mixture F-12 Ham, 0.25% (w/v) bovine serum albumin (BSA), 0.75% D-glucose, 0.25% L-Glutamine, 0.5% B-27 supplement, 0.1% kynurenic acid, 0.01% of 70 % uridine and 30% fluoro-deoxy-uridine.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner</td>
			<td>#60-628160, #60-627160</td>
			<td>60mm, 35mm, respectively.</td>
		</tr>
		<tr>
			<td>Poly D &#8211; Lysine</td>
			<td>Sigma</td>
			<td>#P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Smart Dentin Grinder</td>
			<td>KometaBio</td>
			<td>#GR101</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>#15-090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>#U3750</td>
			<td></td>
		</tr>
	</tbody>
</table>

increasing durability of dissociated neural cell cultures using biologically active coralline matrix

해리된 해마 신경 세포 및 신경교 세포의 배양은 높은 세포 격리와 통제된 환경을 제공함으로써 신경 성장 및 기능을 연구하기 위한 귀중한 실험 모델입니다. 그러나, 시험관 내에서 해마 세포의 생존은 손상된다 : 대부분의 세포는 배양 첫 주 동안 죽는다. 따라서 배양에서 신경 세포의 내구성을 높이는 방법을 식별하는 것이 매우 중요합니다.
산호의 골격에서 유래 한 결정질 아라고나이트 형태의 탄산 칼슘은 신경 배양을위한 우수한 활성 매트릭스로 사용될 수 있습니다. 신경교 세포를 육성, 보호 및 활성화함으로써 산호 골격은 다른 매트릭스보다 시험관 내에서 이러한 세포의 생존과 성장을 더 잘 향상시킵니다.
이 프로토콜은 산호 매트릭스에서 해마 세포를 배양하는 방법을 설명합니다. 이 매트릭스는 산호 골격 알갱이를 배양 접시, 플라스크 및 유리 커버 슬립에 부착하여 생성됩니다. 곡물은 세포를 미세한 3차원(3D) 환경에 도입하여 성장하고 조직과 같은 구조를 형성함으로써 세포의 환경을 개선하는 데 도움이 됩니다. 산호 골격에 의해 도입 된 3D 환경은 분쇄를 통해 세포에 최적화 될 수 있으며, 이는 신경교 세포 활동에 영향을 미치는 것으로 밝혀진 특성 인 입자의 크기와 밀도 (즉, 매트릭스 거칠기)를 제어 할 수 있습니다. 또한, 곡물을 사용하면 특히 광학 현미경을 사용할 때 배양을 더 쉽게 관찰하고 분석할 수 있습니다. 따라서 이 프로토콜에는 시험관 내에서 신경 세포의 유지 및 기능을 개선하기 위한 도구로서 산호 매트릭스의 생성 및 최적화를 위한 절차가 포함됩니다.

산호 골격 매트릭스를 준비하기 위해 전체 산호 골격 (그림 1A)을 망치 (그림 1B)를 사용하여 0.5-2cm 조각으로 나누고 10 % 차아 염소산염 용액, 1M NaOH 용액 및 30 % H 2 O2용액 (그림 1C)을 사용하여 3 단계 (프로토콜의 1 단계)를 통해 유기 잔류 물로부터 철저히 세척했습니다. 산호 조각은 골격 색이 갈색 (?...

Watch this Scientific Journal Video about Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix at JoVE.com

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

해리 된 해마 세포 배양은 신경 과학에서 중추적 인 실험 도구입니다. 산호 골격이 신경 보호 및 신경 조절 역할로 인해 매트릭스로 사용될 때 배양에서 신경 세포 생존과 기능이 향상됩니다. 따라서 산호 기질에서 자란 신경 세포는 더 높은 내구성을 나타내므로 배양에 더 적합합니다.

생물학적 활성 Coralline Matrix를 사용한 해리 된 신경 세포 배양의 내구성 향상

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

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Research

JoVE Journal

Bioengineering

1.2K 조회수.  Ariel University. 해리 된 해마 세포 배양은 신경 과학에서 중추적 인 실험 도구입니다. 산호 골격이 신경 보호 및 신경 조절 역할로 인해 매트릭스로 사용될 때 배양에서 신경 세포 생존과 기능이 향상됩니다. 따라서 산호 기질에서 자란 신경 세포는 더 높은 내구성을 나타내므로 배양에 더 적합합니다.

비디오: Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

심근 조직 공학을위한 생물학 유래 주사용 재료의 제조

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

연구

생체공학

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

활성 세포 배양을위한 형상 기억 폴리머

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

어쿠스틱 Microstreaming를 사용하여 표준 실험실 역방향 Transcriptase 반응에서 mRNA의 단일 세포로부터 cDNA 수량 산출 증가

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

라만 분광학을 사용하여 세포와 세포외 기질의 비 접촉, 레이블이없는 모니터링

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

문화를 셀에 충격파 응용 프로그램

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

생체 외에서 Neuroinflammation의 모델링에 대 한 기본 Glial 세포의 3 차원 하이드로 겔 문화 개선

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

큰 힘줄 결함을 복구하는 조직 엔지니어링 콜라겐 이식과 새로운 테노르래피 봉합사 기술

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

날조는 신장 피 질 세포 외 매트릭스 파생 하이드로 겔

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

희생 빈 섬유 막 세포 배양을 통해 기질 섬유의 생산

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...